Cathepsin S (EC 3.4.22.27) is a cysteine protease of the papain family found primarily in lysosomes (D. Bromme and M. E. McGrath, xe2x80x9cHigh Level Expression and Crystallization of Recombinant Human Cathepsin Sxe2x80x9d, Protein Science (1996) 5:89-791). Cathepsin S (CatS) is a cysteine protease expressed in lymphatic tissues. It is has been identified as playing a major role in invariant chain proteolysis which is a prerequisite for peptide loading of MHC class II (Riese et al., Immunity (1996) 4:357). It has 50 to 60% identity to cathepsins L and K, but differs in that it has a broad pH optimum that extends to alkaline pH. Inhibitors have been shown in animal models to modulate antigen presentation and are effective in an asthma model (Riese et al., J. Clin. Invest. (1998) 101:2351). Mice deficient in cathepsin S have an impaired ability to present exogenous proteins by professional antigen presenting cells (Nakagawa et al., Immunity (1999) 10:207; Shi et al., Immunity (1999) 10:197).
The role of cathepsin S in the immune response is anticipated by its tissue distribution: cathepsin S is found primarily in lymphatic tissues, lymph nodes, the spleen, B lymphocytes, and macrophages (H. Kirschke, xe2x80x9cChapter 211: Cathepsin Sxe2x80x9d in Handbook of Proteolytic Enzymes, (A. J. Barrett, N. D. Rawlings, and J. F. Woessner (Eds.)) San Diego:Academic Press (1998) 621-624). Cathepsin S inhibitors have been shown in animal models to modulate antigen presentation and are effective in an animal model of asthma (R. J. Riese, R. N. Mitchell, J. A. Villadangos, G. P. Shi, J. T. Palmer, E. R. Karp, G. T. De Sanctis, H. L. Ploegh, and H. A. Chapman, xe2x80x9cCathepsin S Activity Regulates Antigen Presentation and Immunityxe2x80x9d, J. Clin. Invest. (1998) 101:2351-2363; G. P. Shi, J. A. Villadangos, G. Dranoff, C. Small, L. Gu, K. J. Haley, R. Riese, H. L. Ploegh, and H. A. Chapman, xe2x80x9cCathepsin S Required for Normal MHC Class II Peptide Loading and Germinal Center Developmentxe2x80x9d, Immunity (1999) 10:197-206).
The recognition of antigen-presenting MHC class II molecules by CD4+ T cells is a crucial component of the immunological response. Class II molecules, like other transmembrane proteins, are translocated into the endoplasmic reticulum after synthesis, where they associate with a third protein, the invariant chain (Ii). This molecule is a type II transmembrane protein that serves as a class II-specific chaperone which promotes the exit of class II-Ii complexes from the endoplasmic reticulum and prevents class II molecules from binding peptides and unfolded proteins in the endoplasmic reticulum and in the secretory pathway.
A targeting motif in the cytoplasmic tail of Ii directs the complexes from the secretory pathway into the endosomal system. Before the MHC class II molecules can present antigen, the Ii must be removed. This is accomplished by a series of proteases that break Ii down into small peptides. However, an Ii fragment, called class II-associated invariant chain peptide (CLIP), which occupies the peptide-binding groove of the class II molecule, is in most cases not spontaneously released. The CLIP fragment serves as a substitute peptide that protects the class II binding pocket from collapsing both during intracellular transport and after Ii degradation in the endosomal system. Binding of antigenic peptides, generated from endocytosed proteins, requires an empty, yet open binding site, and therefore CLIP has to be released while the open binding site needs to be stabilized to allow the binding of other peptides. Human Leukocyte Antigen DM (xe2x80x98HLA-DMxe2x80x99) has been well documented to mediate both of these functions, thus promoting the binding of antigenic peptides. After acquiring peptides, the class II molecules are transported to the cell surface via routes that are largely unknown.
Blocking the presentation of antigens is a promising way to inhibit the immune response. This could be done by disrupting the uptake, the proteolytic processing, or binding to MHC class II molecules. Blocking the uptake may be problematic since many different cell types require this function. Inhibition of the proteolytic processing of particular antigens may be of use since different proteases may be involved in cleaving different antigens, however, these proteases are not specific and may lead to other side effects. One way to specifically block the binding to the antigens to the MHC class II is to inhibit the proteolysis of the invariant chain. If this is not removed, the MHC class II molecules cannot be loaded with peptides, hence blocking Ii degradation would decrease antigen presentation to CD4+ T-cells and disrupt the normal immune response.
Mice in which the gene encoding cathepsin S has been knocked out are less susceptible to collagen-induced arthritis and their immune systems have an impaired ability to respond to antigens (T. Y. Nakagawa, W. H. Brissette, P. D. Lira, R. J. Griffiths, N. Petrushova, J. Stock, J. D. McNeish, S. E. Eastman, E. D. Howard, S. R. M. Clarke, E. F. Rosloniec, E. A. Elliott, and A. Y. Rudensky, xe2x80x9cImpaired Invariant Chain Degradation and Antigen Presentation and Diminished Collagen-Induced Arthritis in Cathepsin S Null Micexe2x80x9d, Immunity (1999) 10:207-217). These data demonstrate that compounds that inhibit the proteolytic activity of human cathepsin S should find utility in the treatment of chronic autoimmune diseases including, but not limited to, lupus, rheumatoid arthritis, and asthma; and have potential utility in modulating the immune response to tissue transplantation.
There are a number of cathepsin S inhibitors reported in the literature, some of which are listed below. Certain dipeptidyl nitrites are claimed by Novartis as cathepsin S inhibitors (Altmann et. al.: WO 99/24460). Dipeptidyl vinyl sulfones are claimed by Arris (now Axys Pharmaceuticals, Inc.) as cysteine protease (including cathepsin S) inhibitors (Palmer et al.: U.S. Pat. No. 5,976,858). Certain peptidyl sulfonamides are claimed by Arris/Axys as cysteine protease (including cathepsin S) inhibitors (Palmer et al.: U.S. Pat. No. 5,776,718 [assigned to Arris, now Axys]; Klaus et al.: U.S. Pat. No. 6,030,946 [assigned to Axys]).
Methods of modulating autoimmunity with an agent that modulates cathepsin S activity, e.g. proteolysis of the Ii chain, as well as methods of treating a subject having an autoimmune disorder, methods of evaluating a treatment for its ability to modulate an immune response are described in WO 99/58153.
A DNA molecule encoding monkey cathepsin S has been cloned and characterized and it represents a novel nucleotide and amino acid sequence. Using a recombinant expression system functional DNA molecules encoding the monkey cathepsin S protease have been isolated. The biological and structural properties of these proteins are disclosed, as is the amino acid and nucleotide sequence. The recombinant DNA molecules, and portions thereof, are useful for isolating homologues of the DNA molecules, identifying and isolating genomic equivalents of the DNA molecules, and identifying, detecting or isolating mutant forms of the DNA molecules. The recombinant protein is useful to identify modulators of functional cathepsin S. Modulators identified in the assays disclosed herein are useful as therapeutic agents. The monkey cathepsin S nucleic acid and polypeptide molecules of the present invention are also useful in studying the properties of cathepsin S modulators, such as in developing compounds intended for pharmaceutical use in any species, including but not limited to humans.